Mapping of a novel locus Ra conferring extreme resistance against potato virus A in cultivated potato (Solanum tuberosum L.).

KEY MESSAGE: The Ra extreme resistance against potato virus A was mapped to the upper of chromosome 4 in tetraploid potato. Potato virus A (PVA) is one of the major viruses affecting potato worldwide and can cause serious disease symptoms and yield losses. Previously, we determined that potato cultivar Barbara harbors Rysto (genotype: Ryryryry) and Ra (genotype: Rararara) that each independently confer extreme resistance to PVA. In this study, employing a combination of next-generation sequencing and bulked-segregant analysis, we further located this novel Ra on chromosome 4 using a tetraploid BC1 potato population derived from a Ry-free progeny (Rararararyryryry) of Barbara (RarararaRyryryry) × F58050 (rararararyryryry). Using 29 insertion-deletion (InDel) markers spanning chromosome 4, Ra was delimited by the InDel markers M8-83 and M10-8 within a genetic interval of 1.46 cM, corresponding to a 1.86-Mb genomic region in the potato DM reference genome. The InDel marker M10-8, which is closely linked with the resistance against PVA in the Ry-free segregating populations, was then used to screen 43 selected Rysto-free tetraploid potato breeding clones. The phenotype to PVA was significantly correlated with the present/absent of the marker, albeit with a 9.3% false positive rate and a 14.0% false negative rate. These findings are of importance in furthering the cloning of Ra and employing the marker-assisted selection for PVA resistance.

Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato.

In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6-98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.

Generation of virus-resistant potato plants by RNA genome targeting.

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA-targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.

RT-PCR Differentiation, Molecular and Pathological Characterization of Andean and Ordinary Strains of Potato virus S in Potatoes in China.

A survey of potatoes in a field in Hubei, China, for common potato viruses revealed that Potato virus S (PVS) was the most abundant virus. To unveil the strain identity of the virus, primers specific to the ordinary and/or Andean strains of PVS (i.e., PVSO and PVSA) were designed. RT-PCR using these primers successfully detected PVSO and PVSA in the samples. Sequence analysis of the amplicons confirmed the correctness of the RT-PCR assay. Two isolates, PVS HB24 and PVS HB7, representing PVSO and PVSA, respectively, were chosen for molecular and biological characterization. Both isolates contained a genome of 8,453 nt in length with six open reading frames. They shared a sequence identity of 79.5% at the complete genome sequence level. Phylogenetic analysis placed PVS HB24 and PVS HB7 to PVSO and PVSA clades, respectively. PVS HB24 induced chlorotic local lesions on the inoculated leaves but no visible symptom on the upper uninoculated leaves of Chenopodium quinoa after mechanical inoculation, whereas PVS HB7 induced both local and systemic symptoms on C. quinoa. ELISA and RT-PCR confirmed that PVS HB7 infected C. quinoa systemically whereas PVS HB24 failed to do so. Both isolates infected potato cv. Shepody and Solanum chacoense asymptomatically, but did not infect Nicotiana occidentalis and N. tobaccum cv. Samsun.

Global Screening and Functional Identification of Major HSPs Involved in PVY Infection in Potato.

HSP40 (also known as DnaJ), HSP70, and HSP90 are major heat shock protein (HSP) families that play critical roles in plant growth and development and stress adaption. Recently, several members of the three HSP families were reported to be widely involved in the plant host-virus interactions. However, their global expression profiles and core members recruited by viruses are largely unknown. In this study, a total of 89 StDnaJs were identified from a genome-wide survey, and their classification, phylogenetic relationships, chromosomal locations, and gene duplication events were further analyzed. Together with 20 StHSP70s and 7 StHSP90s previously identified in the potato genome, the global expression patterns of the members in 3 HSP families were investigated in 2 potato cultivars during Potato virus Y (PVY) infection using RNA-seq data. Of them, 16 genes (including 8 StDnaJs, 6 StHSP70s, and 2 StHSP90s) were significantly up- or downregulated. Further analysis using qRT-PCR demonstrated that 7 of the 16 genes (StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ63, StHSP70-6, StHSP70-19, and StHSP90.5) were remarkably upregulated in the potato cultivar 'Eshu 3' after PVY infection, implying their potential roles in the potato-PVY compatible interaction. Subsequent virus-induced gene silencing (VIGS) assays showed that silencing of the homologous genes of StDnaJ17, StDnaJ21, StHSP70-6, and StHSP90.5 in Nicotiana. benthamiana plants dramatically reduced the accumulation of PVY, which indicated the four genes may function as susceptibility factors in PVY infection. This study provides candidate genes for exploring the mechanism of potato-PVY compatible interaction and benefits breeding work aiming to produce new cultivars with the ability to grow healthily under PVY infection.

Eukaryotic translation initiation factor 4E family member nCBP facilitates the accumulation of TGB-encoding viruses by recognizing the viral coat protein in potato and tobacco.

Due to their limited coding capacity, plant viruses have to depend on various host factors for successful infection of the host. Loss of function of these host factors will result in recessively inherited resistance, and therefore, these host factors are also described as susceptibility genes or recessive resistance genes. Most of the identified recessive resistance genes are members of the eukaryotic translation initiation factors 4E family (eIF4E) and its isoforms. Recently, an eIF4E-type gene, novel cap-binding protein (nCBP), was reported to be associated with the infection of several viruses encoding triple gene block proteins (TGBps) in Arabidopsis. Here, we, for the first time, report that the knockdown of nCBP in potato (StnCBP) compromises the accumulation of potato virus S (PVS) but not that of potato virus M (PVM) and potato virus X (PVX), which are three potato viruses encoding TGBps. Further assays demonstrated that StnCBP interacts with the coat proteins (CPs) of PVS and PVM but not with that of PVX, and substitution of PVS CP in the PVS infectious clone by PVM CP recovered the virus infection in StnCBP-silenced transgenic plants, suggesting that the recognition of PVS CP is crucial for StnCBP-mediated recessive resistance to PVS. Moreover, the knockdown of nCBP in Nicotiana benthamiana (NbnCBP) by virus-induced gene silencing suppressed PVX accumulation but not PVM, while NbnCBP interacted with the CPs of both PVX and PVM. Our results indicate that the nCBP orthologues in potato and tobacco have conserved function as in Arabidopsis in terms of recessive resistance against TGB-encoding viruses, and the interaction between nCBP and the CP of TGB-encoding virus is necessary but not sufficient to determine the function of nCBP as a susceptibility gene.

Susceptibility factor StEXA1 interacts with StnCBP to facilitate potato virus Y accumulation through the stress granule-dependent RNA regulatory pathway in potato.

Plant viruses recruit multiple host factors for translation, replication, and movement in the infection process. The loss-of-function mutation of the susceptibility genes will lead to the loss of susceptibility to viruses, which is referred to as 'recessive resistance'. Essential for potexvirus Accumulation 1 (EXA1) has been identified as a susceptibility gene required for potexvirus, lolavirus, and bacterial and oomycete pathogens. In this study, EXA1 knockdown in potato (StEXA1) was found to confer novel resistance to potato virus Y (PVY, potyvirus) in a strain-specific manner. It significantly compromised PVYO accumulation but not PVYN:O and PVYNTN. Further analysis revealed that StEXA1 is associated with the HC-Pro of PVY through a member of eIF4Es (StnCBP). HC-ProO and HC-ProN, two HC-Pro proteins from PVYO and PVYN, exhibited strong and weak interactions with StnCBP, respectively, due to their different spatial conformation. Moreover, the accumulation of PVYO was mainly dependent on the stress granules (SGs) induced by StEXA1 and StnCBP, whereas PVYN:O and PVYNTN could induce SGs by HC-ProN independently through an unknown mechanism. These results could explain why StEXA1 or StnCBP knockdown conferred resistance to PVYO but not to PVYN:O and PVYNTN. In summary, our results for the first time demonstrate that EXA1 can act as a susceptibility gene for PVY infection. Finally, a hypothetical model was proposed for understanding the mechanism by which StEXA1 interacts with StnCBP to facilitate PVY accumulation in potato through the SG-dependent RNA regulatory pathway.